PROJECT SUMMARY Tuberous sclerosis complex (TSC) is a genetic disorder characterized by the growth of numerous noncancerous (benign) tumors in brain and many other parts of the body. This disease affects as many as 50,000 people in US. TSC often affects brain and the tumors of subependymal giant astrocytoma (SEGA) can cause serious or life-threatening complications. The mutations of Tsc1 or Tsc2 gene leading to the loss of their tumor suppressor functions to control the activity of mTOR underlie the pathogenesis of TSC. mTOR stimulates the activity of S6K and 4EBP1 and is a negative regulator of autophagy. However, recent findings, including ours, revealed a higher autophagy level in TSC1-deficient neurons and TSC1-deficient mammary gland tumor cells under energy stress with high mTOR activity, suggesting unrecognized functions of autophagy in TSC1-deficient cells. To further understand the complex interplay between autophagy and mTOR signaling, we established a novel double conditional knockout (cKO) mouse model to delete TSC1 and FIP200, an essential component in autophagy induction complex, in neural stem cells (NSCs). This model also provides a unique tool to study the mechanism of autophagy in abnormal development and tumorigenesis of SEN/SEGA from TSC-deficient NSCs. Our preliminary findings indicated that FIP200 mediated autophagy was indispensable to maintain the high activity of mTOR and was crucial for the tumorigenesis of SEN/SEGA in TSC1-deficient NSCs. In addition, we found that autophagy was used to sustain high mTOR activity in TSC1-deficient cells under the context of glycolysis inhibition, which is a well-known but not totally clarified phenomenon. These findings form the basis of our hypothesis that autophagy satisfies the high energy demanding in TSC1-deficient NSCs with limited energy source supply to maintain their high mTOR activity and tumorigenesis. In Aim1 of this proposal, we will exam the molecular events to induce autophagy, as well as the mechanism of autophagy to regulate signaling pathways and metabolic activity to maintain high mTOR activity using the in vitro cultured NSCs from TSC1 cKO and TSC1/FIP200 double cKO mice. In Aim2, we will adopt pharmacological methods to inhibit autophagy and glycolysis in TSC1-deficient NSCs to test the feasibility of tumor prevention of SEN/SEGA in TSC1 cKO mice. At the end of these studies, we will expand our knowledge of TSC pathogenesis, identify candidate key signaling pathways and metabolic alterations, and develop new therapeutic concepts for continued investigation into the treatment of an important disorder.